The present invention relates to some new optically active clausenamide derivatives, process of the preparation thereof, a pharmaceutical composition containing the same and their medical use, particularly their use in the respects of hypoxia protective, nootropic and neurodegenerative disease such as cerebral ischemia, Alzheimer disease and vascular dementia.
The average life span in Chinese population is now over 70 years of age.It increased by 100% as compared with before foundation of P.R. China. A scientific of study in abroad estimates that the proportion of the population over 65 years of age will increase to 18.8% by the 2025, when it will just exceed the proportion of children (18.6%). It means that one of 5 population in about 20 years later will be senile. Alzheimer""s disease(AD) and vascular damentia(VD) refer to the presenile and senile form of the disease. i.e. under age 65 vs age 65 and over. Therefore, with the aging of population, the incidence of AD and VD will certainly be increased. The old men and their related neurodegenerative diseases especially various dementia have to undergoing two kinds of death, firstly the psychological death and then physical death. This is a heavy load for not only patients but also families and society. The aging of population is considered as a disadvantage factor, next to the war, pestilence, farming and shortage of resource and energy, to society""s development and stability.
There are many drugs for prevention and treatment of senile dementia, such as cerebral vasodilators which can increase cognition function through improvement of blood flow and energy. However, a real valuable cerebral vasodilator should have high selectivity with no effect on brain metabolism and no xe2x80x9cblood stealxe2x80x9d phenomenon, it needs also to have antiplatelet aggregation and antithrombosis actions. Calcium channel blocker nimodipine accords with some demands as mentioned above, it acts on L-type but not the N- and T-type voltage dependent calcium channel. The drugs for central cholinergic system, the precoursor of acetyecholine(Ach) has weak therapeutic effect. Ach receptor agonists and cholinesterase inhibitors have certain sympmatically therapeutic effect, but its effect is short, and more important is that they can not affect athogenesis of dementia. Many neuropeptides and neurotrophine factors were considered as promising antidementia agents, but its clinical therapeutic effect is not good. The main reason for this is that these large molecules could not pass through the blood-brain barrier. Piracetam or 2-oxo-1 pyrolidine acetamide is usually considered as the prototype nootropic drug. In recent years, clinical studies indicated that the effect of piratcetam in improving memory impairment was mild or obscure. In addition, its mechanism of action is not fully understood, although it has been studied for nearly 30 years.
Racemic clausenamide was isolated from the aqueous extract of leaves of Rutoceae Clausena lausium(Lour) Skeels and had been claimed to have pronounced cerebal hypoxia protective and antiamnestic effect. Its potential medical use and processes of preparation from the plant had been granted a patent right for Chinese Academy of Medical Sciences and Bayer AG(EU patent No. 072514, U.S. Pat. No. 4,879,391 and Chinese patent No. 90107309). Methods for chemical synthesis of racemic clausenamide also had been applied for patent protection (German patent No. 3927370, EP No. 0414120).
The object of the present invention is to provide a new compound for the prevention and/or treatment of neurodegenerative disease such as cerebral ischemia, Alzheimer disease and vascular dementia.
Levoclausenamide as one of the active enantiomer of the racemic clausenamide is generally believed that an optically active clausenamide is the double of that of racemic clausenamide in respect to a biological activity or no any activity. The present inventors have now unexpectedly found that the biological activity of Levoclausenamides is significantly stronger than double of that of racemic clausenmide and is at 5-10 times more potent than that of racemic clausenamide in respect of said activity.
Furthermore, in both behavioral and electrophysiological studies. Levoclausen amide showed to facilitate learning and memory and increase neuronal plasticity. Besides,(xe2x88x92)clausenamide could antagonize Axcex2 induced neurotoxicity, inhibit neuron""s apoptosis and tau protein over phosphorerytion, and therefore (xe2x88x92) clausenamide may be used as drug for prevention and/or treatment of dementia in early stage. The nootropic and antidementia tests of (xe2x88x92) clausenamide have been carried out in biochemical, molecular biological and morphological fields. For example, (xe2x88x92) clausenamide has been indicated to increase synapses of dentate gyrus in adult rats.
The present invention is directed to a new pharmacologically active chiral compound a substituted xcex4-butyrolactan (hereinafter called as levoclausenamide or (xe2x88x92) clausenamide) with the absolute configuration of 3S4R5R6S with levo rotation as shown in formula 1. 
The present invention provides to some new optically active stereoisomeric clausenamide derivatives of formula II, 
wherein said optically active clausenamide derivatives of formula II is selected from one of the following compound:
*3S4R5R6R (xe2x88x92)epiclausenamide,
*3R4S5S6S (+)epiclausenamide
**3S4R5S6R (xe2x88x92)neoclausenamide,
**3R4S5R6S (+)neoclausenamide
**3S4R5S6S(xe2x88x92)epineoclausenamide,
**3R4S5R6R(+)epineoclausen amide
*3S4S5S6R (+)cisclausenamide,
*3R4R5R6S (xe2x88x92)cisclausenamide
*3S4S5S6S (+)cisepiclausenamide,
*3R4R5R6R (xe2x88x92)cisepiclausenamide
*3S4S5R6S (xe2x88x92)cisneoclausenamide,
*3R4R5S6R (+)cisneoclausenamide
*3S4S5R6R(xe2x88x92)cisepineoclausenamide, and
*3R4R5S6S(+)cisepineoclausen-amide
wherein xe2x80x9c*xe2x80x9d indicates new optically active clausenamide compounds;
xe2x80x9c**xe2x80x9d indicates that racemic compounds of which is known but some biological activities thereof concerning noortropic, neurodegenerative disease have not been reported up to now.
The invention provides to some optically active clausenamide derivatives of formula I selected from one of the following compounds for the prevention and/or treatment of neurodegenerative diseases:
3S, 4R, 5R, 6S (xe2x88x92) Levoclausenamide,
3S4R5R6R (xe2x88x92) epiclausenamide,
3R4S5S6S (+)epiclausenamide,
3S4R5S6R (xe2x88x92)neoclausenamide,
3R4S5R6S (+)neoclausenamide,
3S4R5S6S(xe2x88x92)epineoclausenamide,
3R4S5R6R(+)epineoclausen amide,
3S4S5S6R (+)cisclausenamide,
3R4R5R6S (xe2x88x92)cisclausenamide,
3S4S5S6S (+)cisepiclausenamide,
3R4R5R6R (xe2x88x92)cisepiclausenamide,
3S4S5R6S (xe2x88x92)cisneoclausenamide,
3R4R5S6R (+)cisneoclausenamide,
3S4S5R6R(xe2x88x92)cisepineoclausenamide, and
3R4R5S6S(+)cisepineoclausen-amide.
The invention provides to a pharmaceutical composition comprising some optically active compounds of formula I and/or II and pharmaceutically carrier or excipient.
The invention provides to a pharmaceutical composition for the prevention and/or treatment of neurodegenerative diseases comprising any one of the optically active compounds of formula I and pharmaceutically carrier or excipient.
The invention provides to a use of any one of the optically active compounds of formula I and/or II for the manufacture of medicament for the prevention and/or treatment of neurodeganerative diseases.
The invention provides a method for the prevention and/or treatment of neurodegenerative disease comprising administrating to a patient suffered with neurodegenerative disease or high risk of neurodegenrative disease an effective prevention and/or treatment amount of any one of the optically active compounds of formula I and/or II.
The invention provides to a process for the preparation of levoclausenamide, which characterized in that:
1. De novo synthesis by using intramoleuclar inductive asymmetrical Dargen reaction to prepare compound (2*); 
2. Biocatalystic resolution of said compound (2*); or
3. Chemical resolution of the racemic clausenamidone of formula (5*) 
The present invention provides an optically active Levoclausenamide of formula I 
The present invention is directed to optically active clausenamide derivatives of formula I. 
According to this invention, the compounds of formula I in the instant invention are one selected from the following specific compounds:
3S, 4R, 5R, 6S (xe2x88x92) Levoclausenamide,
3S4R5R6R (xe2x88x92) epiclausenamide,
3R4S5S6S (+)epiclausenamide
3S4R5S6R (xe2x88x92)neoclausenamide,
3R4S5R6S (+)neoclausenamide
3S4R5S6S(xe2x88x92)epineoclausenamide,
3R4S5R6R(+)epineoclausen amide
3S4S5S6R (+)cisclausenamide,
3R4R5R6S (xe2x88x92)cisclausenamide
3S4S5S6S (+)cisepiclausenamide,
3R4R5R6R (xe2x88x92)cisepiclausenamide
3S4S5R6S (xe2x88x92)cisneoclausenamide,
3R4R5S6R (+)cisneoclausenamide
3S4S5R6R(xe2x88x92)cisepineoclausenamide, and
3R4R5S6S(+)cisepineoclausen-amide.
The present invention also relates to a pharmaceutical composition comprising an optically active compound of formula I and/or II as an active ingredient and suitable pharmaceutically acceptable carrier or excipient. The an optically active compound of formula I and/or II are administrated alone or in the form of a pharmaceutical composition containing the same. Administration route may be intestinal or parenteral, such as oral, intramuscular, substcutaneous, transdermally, intransally, intraperitoneally, topically etc, said pharmaceutical preparations may formulated by mixing an optically active clausenamide derivatives of formula (I) with pharmaceutically acceptable diluents or excipients.
Furthermore, the pharmaceutical preparations may be various administration form such as a sterile isotonic aqueous solution, tablet, capsule, pill, drop and suppositories, paster, oilment, gel, pastes, cream, spray (including aerosols), lotion, suspensions, solution and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granules or powders.
The diluents to be used in pharmaceutical compositions (e.g. granulates) include customary pharmaceutically acceptable diluents or excipients such as starch, gelatine, silicic acid, polyethylene glycol. For liquid pharmaceutical preparation, a diluent or carrier may be water, alcohol, proplene glycol, vegetable oil, corn oil, peanut oil, olive oil, surface active agents, lubricants, disintegrating agents, preservative agent, flavoring agent or pigment.
According to the present invention, the term xe2x80x9cpatientxe2x80x9d means to mammal, for example, human being.
The term xe2x80x9cneurodegenerative diseasexe2x80x9d includes, for example, cerebral ischemia, Alzheimer disease and vascular dementia etc.
The levoclausenamide [(xe2x88x92)3S, 4R, 5R, 6S]] as one of some optically active clausenamide derivatives of the present invention may be prepared by the de novo asymmetric synthesis and the said detailed synthesis is shown in the following scheme 1. 
Scheme 1 consists of the following steps:
i) treating benzaldehyde with optical alkyl (R) chloroacetate in the presence of basic catalyst to give compound(1),the chiral group from natural or synthetic alcohol with appropriate configuration, preferably (+) menthol, (+) 8-phenylmenthol,(+) 8-xcex2-naphthyl menthol,
ii) adding sodium methoxide to a solution of compound(1) in methanol to yield compound(2*),(+)2S3R methyl 2,3 epoxy cinnamate,
iii) interchanging easter group of compound(2*) with xcex2-phenyl-ethanol-N-methyl amine in the presence of basic catalyst to yield compound (3*), 3S,4R-N-methyl-N-(xcex2-hydroxyl-ethylbenzyl) 2,3 epoxy cinnamate compound(3*), or said compound (3*) is obtained directly with compound 1 by the procedure as described in (iii),
iv) oxidizing compound 3 with potasssium permanganate and cuppric sulfate to afford (+) 3S4R-N-methyl-N-benzoylmethyl-2,3 epoxy cinnamide(4*)
v) treating the above compound 4 with basic catalyst to give the cyclized product (xe2x88x92) clausenamidone(5*), base catalyst may be selected from lithium hydroxide, sodium hydroxide, potassium hydride, lithium diisopropyl amide, butyl lithium, tetraalkyl ammonium hydroxide or trialkyl amine. The solvent in the reaction may be selected from water, menthnol, ethanol, low molecular alkanol, or aqueous alkanol.
vi) reducing compound(5*) with boronhyde class of reducing agent such as lithium boronhydride, sodium boronhydride, lithium tris sec-butyl boron hydride to give (xe2x88x92) 3S, 4R, 5R, 6S levoclausenamide I*.
Note: sign* meaning optically active, R is alkyl.
The Levoclausenamide (prepared by scheme 1) of the present invention may also be prepared by biocatalytic resolution of the starting material racemic methyl 2,3 epoxy cinnamate with hydrolase producing microorganisium to give (+) 2S3R-methyl 2,3 epoxy cinnamate in scheme 2, then repeating the steps iii)-vi) in Scheme 1, thereby obtaining levoclausenamide. 
In scheme 2, The hydrolase producing microorganisium used may be selected from such as fungi (e.g. Aspergillus, sp; Mucor sp; Penicillium, sp;Phizopus, sp), bacteria (e.g. Achromobacter, sp; Alcaligenes, sp; Bacillus, sp; Brevibacterium, sp; Corynebac-terium, sp; Erwinia, sp; Pseudomonas, sp), yeast (e.g. Canadida, sp;Pichia, sp), Rhodotorula, sp; Hansenula, sp) or Actomycetals (e.g. Nocardia, sp; Strep-tomyces, sp). The above mentioned microorganisium may be cultured under aerobic condition in medium containing carbon, nitrogen source with appropriate inducer and inorganic salt at room temperature or heating, preferably at 20-40xc2x0 C., PH5-8,the mycelium produced is collected for stereo-selective hydrolysis of racemic methyl 2,3 epoxy cinnamate as shown in scheme 2.
To the above mentioned culture medium surface active agent such as polyethylene glycol, tween, polyvinyl alcohol, hexadecyl-trimethyl ammonium bromide at concentration of 0.5-5%, preferably 0.5-2% may be added, if necessary.
The stereoselective hydrolysis is proceeded in appropriate organic solvent such as benzene, toluene, xylene, ethyl-ether, ethyl acetate, isopropyl ether, carbon tetrachloride, chloroform preferably toluene, xylene or isopropyl ether, and in such condition that the concentration of substrate as 0.05-10%, preferably 0.5-5%, 10-50xc2x0 C. of temperature, PH 5-10, preferably 6-9.
Furthermore, the above mentioned enzymatic hydrolysis is carried out in aqueous buffer solution or in both of biphasic aqueous buffer and organic solvent such as toluene, benzene, ether, ethyl acetate, isopropyl ether, toluene, carbon tetrachloride or chloroform, preferable toluene, xylene, or isopyl ether.
The isolation of the optical active methyl 2,3 epoxy cinnamate as product is conducted by the known process in the art, such as, extracting the reaction mixture with organic solvent or separating the organic layer in case of biphasic reaction being applied, washing the organic extract or organic layer with saturated sodium bisulfite solution to remove the phenylacetaldehyde, drying, concentrating to give compound 2, (+) 2S3R, mehyl 2,3 epoxycinnamate.
The levoclausenamide (prepared by scheme 1 or scheme 2) of the present invention may also be prepared by resolution of the intermediate, racemic clausenamidone. The details is shown in scheme 3. 
In scheme 3, the resolving agent is selected from chiral acid chloride such as optically active alkyoxy substituted acylhalide, N-phthalyl optical active amino acid halide, or optical active sulfonyl halide, preferably menthoxy active chloride, N-phthalyl (xe2x88x92) alanyl chloride. said resolution comprising following steps:
ixe2x80x2) easterifying racemic clausenamidone with optical active acyl chloride in the presence of pyridine,
iixe2x80x2) separating the distereomers (xe2x88x925*)(+5*) by recrystallization or column chromatography.
iiixe2x80x2) reducing (xe2x88x925*) by sodium borohydride followed by hydrolysis in the presence of NaOH, giving levoclausenamide (xe2x88x92I*)
The present invention is further directed to the method of preparation of optically active clausenamide derivatives of the general formula II selected from one of the following compounds. 
3S4R5S6R (xe2x88x92)neoclausenamide,
3R4S5R6S (+)neoclausenamide
3S4R5S6S(xe2x88x92)epineoclausenamide,
3R4S5R6R(+)epineoclausen amide
3S4S5S6R (+)cisclausenamide,
3R4R5R6S (xe2x88x92)cisclausenamide
3S4S5S6S (+)cisepiclausenamide,
3R4R5R6R (xe2x88x92)cisepiclausenamide
3S4S5R6S (xe2x88x92)cisneoclausenamide,
3R4R5S6R (+)cisneoclausenamide
3S4S5R6R(xe2x88x92)cisepineoclausenamide, and
3R4R5S6S(+)cisepineoclausen-amide.
wherein the process may be summarized in following Scheme 4 
or the process for preparation of epiclausenamide is showed in scheme 5: 
According to the present invention, the following optically active clausenamide derivatives having following physico-chemical features have been obtained.
The present invention is further directed to the facilitating learning and memory activity and inducing long-term potentiation of some optically active clausenamide derivatives. Their nootropic action was 5-10 times and 50-100 times more than that of (xc2x1)-clausenamide and piracetam respectively. For example, (xe2x88x92)clausenamide could also improve spatial memory impairment induced by xcex2-amyloid (A xcex2 25-35). The nootropic mechanisms may be deduced as follows:
1. (xe2x88x92) clausenamide was a potassium channel blocker which induces an increase of intracellular Ca2+ level and then actiate cAMP and PKC to facilitate memory and LTP.
2. (xe2x88x92) clausenamide increased synapses density in weaning mice and mossay fiber sprouting in adult rat hippocampus.
3. (xe2x88x92) clausenamide increased Ach content and ChAT activity in cortex, hippocampus and striatum.
4. (xe2x88x92) clausenamide increased protein biosyntheses of mice brain and promoted zif/268 mRNA and protein expression.
According to the present invention, some new optically active clausenamide derivatives of the invention unexpectedly shows the improvement of hydpoxia, nootropic, neurodegerrerative diseases such as cerebral ischemia, Alzheimer diseases, vascular and antiazonit action of neurocell apoptosis. In particular, for example, (xe2x88x92) clausenamide at concentration of 10xe2x88x927xe2x88x9210xe2x88x925 mol/l inhibited apoptosis significantly In three apoptotic models,. Further studies indicates that the inhibitory of (xe2x88x92) clausenamide on apoptosis may be relevant with its activity in respect of promoting Bcl-2 expression, raising the mitochondrial complex I and complex IV activity and inhibitory release of cytochrome C.